This invention relates to the field of immunology. In particular, it relates to peptides which form complexes with HLA molecules, and exhibit a half-life of more than 30 minutes relative to degradation by peptidase. More particularly, the present invention contemplates a class of peptides of a particular nonapeptide, MZ2-E, which have been modified to resist peptidase degradation. The contemplated peptides are useful in many ways, e.g., as immunogens and as materials which target and bind MHC/HLA molecules.
The study of the recognition or lack of recognition of foreign antigen by a host organism has proceeded in many different directions. Understanding of the field presumes some understanding of both basic immunology, and protein chemistry.
The immune system is constantly at war, fighting viruses, bacteria, and other pathogens that try to invade the body. In this war, it uses a diverse range of effectors in order to deal with the threat to health posed by an equally diverse range of infectious organisms that are prevalent in the environment. For example, T-lymphocytes play a central role in the induction and regulation of the immune response and in the execution of immunological functions. These cells are particularly important in tumor rejection and in fighting viral infections.
However, antigen recognition by the T-lymphocytes is restricted by cell-surface glycoproteins encoded by the highly polymorphic genes of the major histocompatibility complex (MHC) molecules. This discrimination requires a T cell specific for a particular combination of an HLA molecule and a peptide rather than the intact foreign antigen itself. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. Much work has focused on the mechanisms by which proteins are processed into the HLA binding peptide. See, in this regard, Cresswell, Nature 343: 593-594 (1990); Aichinger, et al. Biochemical Society Trans, 23: 657-659, (1995); Fremont et al., Science 257: 919 (1992); and Lanzavecchia, Science 260:937-943 (1993); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992).
MHC class I molecules are expressed by almost all nucleated cells of the body and, in the main, present peptides derived from intracellular proteins to cytotoxic T cells expressing the CD8 co-receptor. Activation of the cytotoxic T cell, in turn, results in the destruction of the target cells by apoptosis induced by perforin/granzyme and/or Fas ligand.
In the case of MHC class I molecules, the peptide fragments usually contain from 8 to 11 amino acids and are generated inside the antigen presenting cells by a complex machinery involving proteases, peptide transporters and molecular chaperones. See Roitt, et al. Immunology (Mosby-Year Book Europe, 1993).
Although intact proteins need to be processed to generate antigenic peptide, soluble peptides are also known to directly bind to small fractions of empty MHC class I or II molecules present on cell surfaces. See in this regard, Braciale and Braciale, Immunology Today, 12(4): 124-129 (1991); Elliot, Immunology Today 12(11): 386-388 (1991).
MHC class II molecules are expressed on a more limited set of antigen presenting cells, exemplified by B-cells, T-cells themselves, macrophages, and dendritic cells. These molecules present peptides in a way which is similar to class I molecules, except that the peptide is generally derived from an exogenous protein from the intercellular environment (e.g., bacteria). Class II molecules present their captured peptide to helper T cells expressing the CD4 co-receptor molecule and their activation generally results in the secretion of cytokines.
Thus, specific T cell immunity is controlled by two selective and independent binding events: first, binding of the peptide fragments of the antigens by the MHC class molecules, and second, binding of the resulting complexes by the clonotypic antigen receptors of the T cell. See, in this regard, Ada, Immunology and Cell Biology 72:447-454 (1994).
Tumor antigens are characteristic of tumor tissue and thus may be considered tissue specific. Tumor antigens result from alterations that frequently occur in malignant transformation of normal tissue. The alteration may be quantitative in that a particular normal antigen may decrease or increase in concentration. Such normal antigens that have increased concentration in tumors are generally referred to as xe2x80x9cTumor Associated Antigens (TAA).xe2x80x9d Antigenic alteration may also be qualitative in that a new antigen, foreign to the host, may appear. These are termed xe2x80x9cTumor-Specific Antigens (TSA)xe2x80x9d and may be present as new cell-surface structures or as new intracellular structures in the cytoplasm or nucleus.
Tumor specific antigens were first clearly demonstrated in mice that had been immunized with cells from a methylcholanthrene-induced sarcoma taken from syngeneic mice. These molecules were xe2x80x9crecognizedxe2x80x9d by T cells in the recipient animal, and provoked a cytolytic T cell (xe2x80x9cCTLxe2x80x9d hereafter) response with lysis of the transplanted cells. The antigens expressed by the tumors and which elicited the T cell response were found to be different for each tumor. See Prehn, et al., J. Natl. Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30: 2458-2462 (1970) for general teachings on inducing tumors with chemical carcinogens and differences in cell surface antigens. This class of antigens has come to be known as xe2x80x9cTumor Specific Transplantation Antigensxe2x80x9d or xe2x80x9cTSTAsxe2x80x9d. Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974) Prehn, R. T., and Main, J. M, Journal of Natl. Cancer Inst. 18:769 (1974).
While T cell mediated immune responses were observed for the types of tumor described supra, spontaneous tumors were thought to be generally non-immunogenic. These were therefore believed not to present antigens which provoked a response to the tumor in the tumor carrying subject. See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).
The family of tumxe2x88x92 antigen presenting cell lines are immunogenic variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, tumxe2x88x92 antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., xe2x80x9ctum+xe2x80x9d cells). When these tum+ cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus xe2x80x9ctumxe2x88x92xe2x80x9d). See Boon et al., Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which is incorporated by reference. Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).
It appears that tumxe2x88x92 variants fail to form progressive tumors because they elicit an immune rejection process. The evidence in favor of this hypothesis includes the ability of xe2x80x9ctumxe2x88x92xe2x80x9d variants of tumors, i.e., those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl, Acad. Sci. USA 76: 5282-5285 (1979); and the observation that intraperitoneally injected tumxe2x88x92 cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophage (Uyttenhove et al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory which permits them to resist subsequent challenge to the same tumxe2x88x92 variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).
Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor. See Van Pel et al., J. Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it is possible to elicit presentation of a so-called xe2x80x9cTRAxe2x80x9d in a tumor which is a target for a syngeneic rejection response. Similar results have been obtained when foreign genes have been transfected into spontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988) in this regard.
The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies, in vitro i.e., the study of the identification of the antigen by a particular cytolytic T cells (xe2x80x9cCTLxe2x80x9d hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the antigen are lysed.
Characterization studies have identified CTL clones which specifically lyse cells expressing the antigens. Examples of this work may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc. Res. 47: 5074-5079 (1987).
A tumor exemplary of the subject matter described supra is known as P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al., J. Exp. Med. 172: 35-45 (1990), the disclosures of which are incorporated by reference. The P815 tumor is a mastocytoma, induced in a DBA/2 mouse with methylcholanthrene and cultured as both an in vitro tumor and a cell line. The P815 line has generated many tumxe2x88x92 variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra, and P198 (Sibille, supra).
Hence, with reference to the literature, a cell line can be tum+, such as the line referred to as xe2x80x9cP1xe2x80x9d, and can be provoked to produce tumxe2x88x92variants. Since the tumxe2x88x92 phenotype differs from that of the parent cell line, one expects a difference in the DNA of tumxe2x88x92 cell lines as compared to their tum+ parental lines, and this difference can be exploited to locate the gene of interest in tumxe2x88x92 cells. As a result, it was found that genes of tumxe2x88x92 variants such as P91A, 35B and P198 differ from their normal alleles by point mutations in the coding regions of the gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58: 293-303 (1989). This has proved not to be the case with the xe2x80x9ctumor rejection antigensxe2x80x9d or xe2x80x9cTRAsxe2x80x9d of this invention. These papers also demonstrated that peptide derived from the tumxe2x88x92 antigen are presented by the H-2 molecule for recognition by CTLs. P91A is presented by Ld, P35 by Dd and P198 by Kd.
Human melanoma cells also bear antigens that are recognized by autologous CD8+ cytolytic T cells, which can be derived from blood lymphocytes or from tumor-infiltrating lymphocytes. In PCT application PCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, and incorporated by reference, a family of genes is disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific cytolytic T lymphocytes. The genes are said to code for xe2x80x9ctumor rejection antigen precursorsxe2x80x9d or xe2x80x9cTRAPxe2x80x9d molecules, and the peptides derived therefrom are referred to as xe2x80x9ctumor rejection antigensxe2x80x9d or xe2x80x9cTRAsxe2x80x9d. See Traversari et al., Inmmunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on this family of genes. Also, see U.S. Pat. No. 5,342,774, incorporated by reference in its entirety, which discloses the xe2x80x9cMAGExe2x80x9d family of tumor rejection antigen precursors.
The tumxe2x88x92 antigens are only present after the tumor cells are mutagenized. In contrast, tumor rejection antigens-and this is a key distinction-are present on cells of a given tumor without mutagenesis.
U.S. Pat. No. 5,405,940, the disclosure of which is incorporated by reference, contemplates isolated nonapeptides derived from MAGE genes. In this patent, it is explained that the MAGE-1 gene codes for a TRAP which is processed to nonapeptides that are presented by HLA-A1 molecules. According to this patent, the nonapeptides are derived from expression products of the MAGE gene family. The resulting complexes are identified by cytolytic T cells, which can be used in diagnostics or therapeutically. The nonapeptides which bind to HLA-A1 follow a xe2x80x9crulexe2x80x9d for binding in that a motif is satisfied. In this regard, see e.g. PCT/US93/07421; Falk et al., Nature 351: 290-296 (1991); Engelhard, Ann Rev. Immunol. 12: 181-207 (1994); Ruppert et al., Cell 74: 929-937 (1993); Rxc3x6tzschke et al., Nature 348: 252-254 (1990); Bjorkman et al., Nature 329: 512-518 (1987); Traversari et al., J. Exp. Med. 176: 1453-1457 (1992). These references teach that given the known specificity of particular peptide for particular HLA molecules, one should expect a particular peptide to bind to at least one HLA molecule.
A cursory review of the development of the field may be found in Barinaga, xe2x80x9cGetting Some xe2x80x98Backbonexe2x80x99: How MHC Binds Peptidexe2x80x9d, Science 257: 880 (1992); also, see Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992). These papers generally point to a preference that the peptide which binds to an MHC/HLA molecule be nine amino acids long (a xe2x80x9cnonapeptidexe2x80x9d), and to the importance of two so-called anchor residues (most commonly the second and ninth residues of the nonapeptide).
Studies on the MAGE family of genes have now revealed that particular peptides are in fact presented on the surface of tumor cells, and that the presentation of the peptide requires that the presenting molecule be a specific HLA molecule. Complexes of the MAGE-1 tumor rejection antigen (the xe2x80x9cTRAxe2x80x9d or xe2x80x9cnonapeptidexe2x80x9d) and the HLA leads to lysis of the cell presenting it by CTL. This observation has both diagnostic and therapeutic implications, as discussed herein.
It has also been found that, when comparing homologous regions of various MAGE genes to the region of the MAGE-1 gene coding for the relevant nonapeptide, there is a great deal of homology. Homologous peptides can be used for various purposes which includes their use as immunogens, either alone or coupled to carrier peptide. The peptides are of sufficient size to constitute an antigenic epitope, and the antibodies generated thereto may then be used to identify the peptide, either as it exists alone, or as part of a larger polypeptide.
The nonapeptides may also be used as agents to identify various HLA subtypes on the surface of tumor cells, such as melanomas. Via this ability they may serve either as diagnostic markers, or as therapeutic agents. These features are discussed infra.
A second class of antigens represents differentiation antigens encoded by genes that are expressed in melanoma and in normal melanoctytes. Antigens derived from tyrosinase are exemplary of this class.
It is noteworthy that while the prior art peptides might arguably find use as therapeutics or in diagnostics, their contemplated use is short lived in view of their rapid degradation by peptidase activity.
It is an object of the present invention to provide isolated peptides having from 8-11 amino acids which are characterized as binding to an HLA molecule and having a half-life greater than 30 minutes, relative to degradation by peptidase.
It is an object of the present invention to provide peptides based on the MZ2-E antigen (see infra) which target and complex with HLA molecules. Recognition of the peptide analogue/HLA complex by cytolytic T lymphocytes, leads to lysis of the cells presenting the complex. This recognition may be used in diagnostics, or therapeutically.
The above object is achieved by structurally modified analogues of the tumor antigen MZ2-E.
A still further object of the present invention contemplates a method for treating a subject with a pathological condition which includes administering to the subject an amount of the peptide according to the invention, sufficient to alleviate the pathological condition. The administration of the peptide to the subject alleviates the pathological condition by provoking an effector cytolytic T cell response against the pathological condition.
A still further object of the invention contemplates a method for provoking proliferation of a CTL, comprising contacting a cell which presents a complex of the peptide analogues of the invention and an HLA molecule on its surface to a CTL, under conditions favoring proliferation thereof.
An alternative embodiment of the present invention relates to antibodies against the peptide analogues of the present invention.
Another embodiment of the present invention is directed to vaccines useful for treating a pathological disorder characterized by presentation on surfaces of pathological cell of HLA/peptide complexes of a type that differ from normal, wherein the vaccine includes the peptide analogues of the present invention together with a pharmaceutically acceptable adjuvant, which, when administered to a subject, provokes an immune response in the subject.
An alternative embodiment contemplates a method for determining presence of a substance in a sample which binds to a complex of an MHC molecule and a peptide, comprising contacting the sample with (i) a cell which presents said MHC molecule, and (ii) a peptide which consists of an amino acid sequence which specifically complexes with the MHC molecule, wherein the peptide has been modified to render it stable to peptidase degradation for at least 30 minutes, and (iii) determining interaction between said complex and said substance as a determination of the substance in said sample.
A still further embodiment emraces an immunogenic composition comprising the peptide of claim 1, and a pharmaceutically acceptable adjuvant.
The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings, in which like reference numerals designate the same elements.